Analysis Of The Intestinal Microflora In Hepialus Gonggaensis Larvae

Hepialus gonggaensis larvae are predominant host of Cordyceps sinensis. After being parasited, H. gonggaensis become Dongchongxiacao, which is famous and expensive traditional herbal medicine in China and Asia area. There had a few studies on the intestinal microflora of the insects which live in the high altitude and cold area. The present study will be helpful to further understand the significance of different microbial species to the H. gonggaensis larvae.Gut microbial diversity provides insight into the basic function of a gut microbial ecosystem. In this study, H. gonggaensis larvae were collected from mountain Yajiageng of Kangding County, Sichuan province. Hepialus gonggaensis larval guts were first investigated with traditional culturing and culture-independent methods. As been reported previous, only 8 different bacteria of genera had been identified using traditional culturing method. And there are 3 fungi of genera population were identified as Cryptococcus magnus strain, Geomyces sp, Trichosporon porosum strain using traditional culturing method from the guts of H. gonggaensis larvae.Restriction fragment length polymorphism (RFLP) method was used to detection the guts microbiota diversity of H. gonggaensis larvae based on the 16 S rRNA (for bacteria) and ITS (for fungi). 16S rRNA and ITS clone librarys were constructed by extracted total DNA of microorganisms from the guts of H. gonggaensis larvae, amplified 16S rRNA using bacterial universal primers and amplified ITS using fungi-universal primers. The 16S rRNA fingerprints of the microorganisms were analyzed by isolated plasmid and digested them with EcoRI, MspI and Haeâ…¢enzymes respectively. But the ITS fingerprints of the microorganisms were analyzed by isolated plasmid and digested them with MspI, Haeâ…¢and TaqI enzymes respectively.The phylogenetic tree depicted the linkage of the isolated microbial from the guts of H. gonggaensis larvae. The results indicated that the 16S rRNA library established includes 35 restriction endonuclease types. The dominant bacteria in the guts of H. gonggaensis larvae belonged to Rahnella sp and Carnobacterium sp and accounted for 45.58 % and 30.88 % of the total 16S rRNA clones, respectively. The ITS library established include 23 restriction endonuclease types, the dominant fungus in the guts of H. gonggaensis larvae belonged to Mortierella sp. and Trichosporon sp. and accounted for 46.34 % and 39.02 % of the total ITS library, respectively. The results showed the rich microbiota diversity existed in the intestines of H. gonggaensis larvae and microbiota diversity in the guts of H. gonggaensis larvae is remarkable difference from normal environment. There are a lot of microorganisms in the intestines of H. gonggaensis larvae can not be cultured and isolated by tridational method. Therefore more information of microorganism diversity can be obtained by combining the molecular methods based on the 16S rRNA and ITS sequences with traditional culturing methods.